Diagnostic methods for age related macular degeneration

ABSTRACT

Diagnostic methods for identifying a test subject who has or is at risk of developing age-related macular degeneration (AMD) or an analogous disease associated with oxidation of DHA-containing lipids are provided. In one aspect, the methods comprise: assaying for the presence of elevated levels of 2-(ω-carboxyethyl) pyrrole (CEP) adducts in a bodily fluid which has been obtained from the test subject. In a preferred embodiment, such methods comprise providing an antibody that is immunospecific for CEP, contacting a bodily fluid from the subject with the anti-CEP antibody, and assaying for the formation of a complex between the antibody and an antigen in the sample. In another aspect, the methods comprise assaying for the presence of elevated levels of an antibody that binds to or is immunospecific for a CEP adduct in the bodily fluid of the test subject. The present invention also relates to CEP protein and peptide adducts, an antibody reactive with a CEP adduct and a diagnostic kit comprising such antibody.

[0001] This application which claims priority from U.S. ProvisionalApplication Ser. No. 60/287,543, filed on Apr. 30, 2001. This work wassupported, at least in part, by grants GM21249 and HL53315 from theNational Institutes of Health. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0002] This invention relates to novel methods for diagnosing andscreening for age-related macular degeneration and analogous diseasesassociated with docosahexaenoic acid (DHA) containing lipids. Morespecifically, this invention relates to diagnostic methods fordetermining if an individual has or is at risk of developing age-relatedmacular degeneration and atherosclerosis.

[0003] Macular degeneration is the clinical term used to describe thosediseases that are characterized by a breakdown of the macula, the smallportion of the retina responsible for central vision. Juvenile maculardegeneration, also referred to as early onset macular degenerationoccurs early in life, such as for example in the second and thirddecade, while age-related macular degeneration (AMD) occurs later inlife, typically in the fifth decade and later. AMD constitutes a majorhealth problem for individuals over 55 years of age in theindustrialized world. In the USA alone between 6 and 10 million senioradults are legally blind from AMD.

[0004] It is desirable to have diagnostic methods for determining if anindividual has a predisposition for developing age-related maculardegeneration and other diseases which involve oxidative damage totissues from oxidation of DHA-containing lipids.

SUMMARY OF THE INVENTION

[0005] In accordance with the present invention, diagnostic methods foridentifying a test subject who has or is at risk of developingage-related macular degeneration (AMD) or an analogous diseaseassociated with oxidation of DHA-containing lipids are provided. In oneaspect, the methods comprise: assaying for the presence of elevatedlevels of 2-(ω-carboxyethyl) pyrrole (CEP) adducts in a bodily fluidwhich has been obtained from the test subject. As used herein the term“CEP adduct” refers to a molecule which comprises CEP bound to a carriercomprising a primary amino group. Examples of such carriers are aprotein, an amino phospholipid, an amino sugar, an amino acid,particularly lysine, or a metabolic product of these molecules. As usedherein, the term “test subject” refers to a mammal, preferably a human.In a preferred embodiment, such methods comprise providing an antibodythat is immunospecific for CEP, contacting a bodily fluid from thesubject with the anti-CEP antibody, and assaying for the formation of acomplex between the antibody and an antigen in the sample. In anotheraspect, the methods comprise assaying for the presence of elevatedlevels of an antibody that binds to or is immunospecific for a CEPadduct in the bodily fluid of the test subject. Preferably, the level ofone or both of the CEP-related diagnostic markers, i.e., the CEP adductand the anti-CEP antibody, is determined in a bodily fluid obtained fromthe test subject and compared to the level of the diagnostic marker in acorresponding bodily fluid from normal healthy subjects.

[0006] In another aspect, the present invention comprises methods formonitoring progression of AMD or atherosclerosis in a test subject whois known to have AMD or atherosclerosis. Such methods comprisedetermining the levels of CEP adducts, anti-CEP antibodies, or both inbodily fluids taken from the test subject over successive timeintervals. The levels of the CEP adducts or anti-CEP antibodies in thesamples are compared to determine the prognosis of the disease in thesubject. An increase in the levels of the diagnostic marker in a bodilyfluid obtained from the test subject over time is indicative ofincreased oxidative damage to tissues from oxidation of DHA and a poorprognosis.

[0007] In another aspect, the present methods are used to monitor theresponse of the test subject to treatment with a therapeutic compositiontargeted at AMD, atherosclerosis, or another disease associated withoxidative damage to tissues from oxidation of DHA. Such methods comprisedetermining the levels of a CEP adduct, or anti-CEP antibody, or both ina bodily fluid obtained from the test subject before and after suchtreatment. Preferably, the concentration or content of one or both ofthese diagnostic markers is measured in samples taken over successivetime intervals following treatment. A decrease in the levels of one orboth of these markers following administration of an anti-AMD drug tothe subject is indicative of decreased potential for oxidative damage toocular tissues of the subject.

[0008] The present invention also relates to an antibody reactive with aCEP adduct and a diagnostic kit comprising such antibody. The presentinvention also relates to CEP protein and peptide adducts which areuseful for assessing the levels of anti-CEP antibodies in a bodily fluidobtained from a test subject. The present invention also relates tomethods of producing antibodies immuno-specific for CEP adducts and tomethods of producing CEP protein and peptide adducts.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1. Generation of 2-(ω-carboxyalkyl)pyrrole epitopes.

[0010]FIG. 2. Serum anti-CEP antibody titer (λ) in New Zealand whiterabbit immunized with CEP-KLH using CEP-BSA as coating agent.

[0011]FIG. 3. (A) Inhibition curve for binding of anti-CEP-KLH toCEP-BSA by CEP-HSA (λ), CPP-HSA (♦), CHP-HSA (σ), PP-ACA-BSA (ν), andHSA (Δ). (B) Inhibition curve for binding of anti-CEP-KLH to CEP-BSA byCEP-HSA (O) and for binding of anti-CEP-KLH to CEP-GPDH by CEP-HSA (λ),CPP-HSA (σ), CHP-HSA (ν), PP-ACA-BSA (♦) and GPDH (Δ).

[0012]FIG. 4. Inhibition curves for binding of anti-CPP-KLH to CPP-BSAby CPP-HSA (), CEP-HSA (♦), CHP-HSA (□)

[0013]FIG. 5. Characterization of CEP-HSA by MALDI-TOF and tandem MS.(A) MALDI-TOF mass spectrum of tryptic digest of CEP-HSA. Arrows denoteinternal standards. Dots denote tryptic peptides from HSA identified bya “MS-Fit” sequence database search. Asterisks denote possible CEPmodified peptides based on the sum of the adduct mass (122.0366) and themass of the peptide. (B) Nanoelectrospray MS/MS spectrum of the doublycharged ion m/z 571 (from singly charged m/z 1141.6302 in panel A). (C)Nanoelectrospray MS/MS spectrum of the doubly charged ion m/z 1101 (fromsingly charged m/z 2021.0678 in panel A).

[0014]FIG. 6. Inhibition curve for binding of anti-CEP-KLH to CEP-BSA byCEP-HSA () and (A) products from reaction of HOHA-PC with HSA for 1 h(♦), 8 h (▴), 24 h (▪) at 37° C. before hydrolysis; (B) products fromreaction of HOHA-PC with HSA for 1 h (▴), 8 h (▪), 24 h (♦) at 37° C.after hydrolysis.

[0015]FIG. 7. Inhibition curve for binding of anti-CEP-KLH to CEP-BSA byCEP-HSA () and products from oxidation of DHA-PC in the presence of HSAfor 1 h (▴), 8 h (♦), 24 h (□) at 37° C. after hydrolysis.

[0016]FIG. 8. CEP immunoreactivity in the mouse retina.Immunohistochemical staining of retina is prominent in thephotoreceptor/retinal pigment epithelium (RPE) complex. In thephotoreceptor layer, the outer segments (OS) are intensely stained whilethe photoreceptor inner segments (IS) are unlabeled. Less intensestaining is also evident in the inner plexiform layer (IPL). Littlestaining is seen in the OLM (outer limiting membrane), ONL (outernuclear layer), ONL (outer nuclear layer), OPL (outer plexiform layer),INL (inner nuclear layer), or GCL (ganglion cell layer).

[0017]FIG. 9. Inhibition curves for binding of anti-CEP-KLH to CEP-BSAby CEP-HSA (), plasma from an AMD patient (□), and a normal control(▴).

[0018]FIG. 10. Levels of CEP adduct immunoreactivity detected in humanplasma from older volunteers who do not have AMD, (▴) N(83), youngerhealthy volunteers, (□) N(27), and patients who were diagnosed to haveage-related macular degeneration, (♦) AMD. The figure also shows meanlevels detected (O). The error bars indicate the standard deviation(S.D.) for each data set.

[0019]FIG. 11. ELISA of anti-CEP IgG autoantibodies in human plasma.Each bar represents the mean value +/−S.D. of at least 3 independentstudies. The horizontal solid line represents the mean value for theN(83) cohort. The dashed line represents the mean value of the N (83)cohort plus 1 S.D. Bars marked with stars indicate that 7 AMD plasmasconsistently exhibit high antiCEP IgG reactivity in both ELISA anddot-blot (data not shown) analyses, whereas, only 1 control exhibitedhigh antiCEP IgG reactivity in both analyses.

[0020]FIG. 12. Structures of 2-(ω-carboxyethyl)pyrrole, CEP (top), and2-(ω-carboxypropyl)pyrrole, CPP (bottom).

[0021]FIG. 13. Levels of CEP adduct immunoreactivity detected in humanplasma from (♦) 83 aged normal volunteers, (σ) 27 normal young healthyvolunteers, and (ν) 10 patients who were diagnosed to haveatherosclerosis (AS). The figure also shows mean levels detected (O).The error bars indicate the standard deviation (S.D.) for data set.

[0022]FIG. 14. Synthesis of Protein and Peptide Derivatives of DOHA.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Diagnostic methods for identifying test subjects who have or areat risk of developing age-related macular degeneration, atherosclerosis,or an analogous disease which involves oxidative damage to tissues fromoxidation of docosahexaenoic acid (DHA) containing lipids are provided.The methods are based, at least in part, on the discovery that patientsdiagnosed with AMD or atherosclerosis have higher levels of CEP adductsin their blood than normal control subjects. The present methods arealso based, at least in part, on the discovery that patients diagnosedwith AMD have higher levels of anti-CEP antibodies in their blood thannormal healthy subjects.

[0024] In one aspect, the methods comprise assaying for the presence ofelevated levels of CEP adduct in a bodily fluid taken from the testsubject. Preferably, the assay employs an antibody which isimmunospecific for the CEP adduct. In another aspect, the methodscomprise assaying for the presence of elevated levels of antibodieswhich are immunospecific for CEP adducts in a bodily fluid obtained fromthe test subject. Advantageously, the present methods are minimallyinvasive and provide an objective and quantifiable index of ongoing DHAoxidative damage in a test subject.

[0025] The present invention also relates to kits and reagents fordiagnosing diseases which involve oxidative damage to tissues fromoxidation of docosahexaenoic acid (DHA)-containing lipids.

[0026] I. Determining the Levels of CEP Adducts in Bodily Fluids of theTest Subject

[0027] The bodily fluids which may be obtained from the subject and usedas test samples in the present diagnostic methods include blood, serum,plasma, spinal fluid, ocular fluid, and tears. The preferred samples areblood, serum, and plasma. Preferably, an antioxidant and a proteaseinhibitor are added to the test sample immediately after the bodilyfluid is obtained from the subject to prevent spurious oxidation ofDHA-containing lipids or proteolysis of CEP-containing proteins in thetest sample. In those cases where the test sample is to be stored priorto analysis it is preferred that the sample containing the antioxidantand protease inhibitor is quench frozen and stored at approximately −80°C.

[0028] Preferably, the presence of the CEP adduct in the bodily fluid isdetected using an antibody that is immuno-specific for CEP adducts. Theterm “immuno-specific” means the antibodies have at least 100 timesgreater affinity for a CEP adduct than for other2-(ω-carboxyalkyl)pyrrole (CAP) adducts, including2-(ω-carboxypropyl)pyrrole. (CPP) adducts. Such antibodies include, butare not limited to, polyclonal, monoclonal, chimeric, single chain, andFab fragments. Optionally, the diagnosis is made by comparing theamount, concentration or content of CEP in a sample obtained from thetest subject to the titer of CEP in samples obtained from subjectslacking the disease, i.e., healthy or normal subjects. Alternatively,the amount, concentration or content of CEP in the sample may becompared to the amount, concentration or content in correspondingsamples which were taken from the test subject for the purpose ofdetermining baseline concentrations of the CEP, such as for exampleduring an early screening procedure.

[0029] Since the bodily fluids of test subjects contain elevated levelsof both the free acid form of CEP adducts, which are detectable withanti-CEP antibodies, and ester forms of the CEP adducts (e.g., esters ofPC), which are normally not detectable with anti-CEP antibodies, thebodily fluids may be treated with a base (e.g. KOH) prior to contactingthe test sample with the anti-CEP antibody. Such treatment converts CEPesters to CEP free acids, making it possible to detect total alterationsin the levels of all CEP adducts in the bodily fluid of the testsubject, including those present in the form of esters.

[0030] Methods of Determining Levels of CEP Adducts in a Bodily FluidObtained from the Test Subject.

[0031] The levels of CEP adducts in a bodily fluid obtained from thetest subject can be determined using polyclonal or monoclonal antibodiesthat are reactive with a CEP adduct. Anti-CEP-antibodies may be made andlabeled using standard procedures and then employed in immunoassays todetect the presence of phospholipid-bound, amino acid-bound,peptide-bound or protein-bound CEP in the sample. Suitable immunoassaysinclude, by way of example, radioimmunoassays, both solid and liquidphase, fluorescence-linked assays or enzyme-linked immunosorbent assaysand Western blot analysis. Optionally, the immunoassays are also used toquantify the amount of CEP adducts that are present in the sample.

[0032] Antibodies raised against CEP adducts for use in suchimmunoassays are produced by conjugating CEP to a carrier protein orgenerating CEP by modification of a carrier protein with a dioxo fattyacid and then using the adduct to immunize a host animal. Suitable hostanimals, include, but are not limited to, rabbits, mice, rats, goats,and guinea pigs. Various adjuvants may be used to increase theimmunological response in the host animal. The adjuvant used depends, atleast in part, on the host species. For example, guinea pig albumin iscommonly used as a carrier for immunizations in guinea pigs. Suchanimals produce heterogenous populations of antibody molecules, whichare referred to as polyclonal antibodies and which may be derived fromthe sera of the immunized animals.

[0033] Monoclonal antibodies, which are homogenous populations of anantibody that binds to a particular antigen, are obtained fromcontinuous cells lines. Conventional techniques for producing monoclonalantibodies are the hybridoma technique of Kohler and Millstein (Nature356:495-497 (1975)) and the human B-cell hybridoma technique of Kosboret al (Immunology Today 4:72 (1983)). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any classthereof. To increase the likelihood that monoclonal antibodies specificto the CEP are produced, the CEP may be conjugated to a carrier proteinwhich is present in the animal immunized.

[0034] Methods of Determining the Levels of Anti-CEP Antibodies in aBodily Fluid Obtained from the Test Subject.

[0035] The present invention also provides diagnostic methods whichinvolve determining the levels of anti-CEP antibodies in a bodily fluidfrom the subject. The method comprises the steps of contacting thebodily fluid with a CEP peptide or protein adduct, and assaying for theformation of a complex between the CEP peptide or protein adduct andantibodies in the sample. For ease of detection, it is preferred thatthe CEP peptide or protein adduct be attached to a substrate such as acolumn, plastic dish, matrix, or membrane, such as nitrocellulose orpolyvinyl difluoride (PVDF). The sample may be untreated, subjected toprecipitation, fractionation, separation, or purification beforecombining with the CEP peptide or protein adduct. Interactions betweenantibodies in the sample and the CEP peptide or protein adduct aredetected by radiometric, colorimetric, or fluorometric means,size-separation, or precipitation. Preferably, detection of theantibody-CEP peptide or protein adduct complex is by addition of asecondary antibody that is coupled to a detectable tag, such as forexample, an enzyme, fluorophore, or chromophore. Formation of thecomplex is indicative of the presence of anti-CEP antibodies, either IgMor IgG, in the test sample.

[0036] Preferably, the method employs an enzyme-linked immunosorbentassay (ELISA) or a Western immunoblot procedure. Such methods arerelatively simple to perform and do not require special equipment aslong as membrane strips are coated with a high quality antigen

[0037] Comparison of Levels of CEP Adducts or Anti-CEP Antibodies inBodily Fluids Obtaind from a Test Subject to Predetermined Values.

[0038] The levels of each diagnostic marker, i.e., the CEP adducts andthe anti-CEP antibodies, in the test subject's bodily fluid may becompared to a single predetermined value or to a range of predeterminedvalues. If the level of the present diagnostic marker in the testsubject's bodily fluid is greater than the predetermined value or rangeof predetermined values, the test subject has or is at greater risk ofdeveloping or having a disease associated with oxidation ofDHA-containing lipids, e.g. AMD or atherosclerosis, than individualswith levels comparable to or below the predetermined value orpredetermined range of values. The extent of the difference between thelevel of the diagnostic marker in the bodily fluid obtained from thetest subject and the predetermined value is also useful forcharacterizing the extent of the disease or risk of developing thedisease and thereby, determining which individuals would most greatlybenefit from certain aggressive therapies. The present diagnostic testsare useful for determining if and when therapeutic agents which aretargeted at preventing AMD or atherosclerosis should and should not beprescribed for a patient.

[0039] Evaluation of Therapeutic Agents Targeted at AMD orAtherosclerosis

[0040] The present diagnostic tests are also useful for evaluating theeffect of therapeutic agents targeted at AMD or atherosclerosis onpatients who have been diagnosed as having or being at risk ofdeveloping AMD or atherosclerosis. Such evaluation comprises determiningthe levels of one or more of the present diagnostic markers includingCEP adducts, anti-CEP antibodies and combinations thereof, in a bodilyfluid taken from the subject prior to administration of the therapeuticagent and a corresponding bodily fluid taken from the subject followingadministration of the therapeutic agent. A decrease in the level of theselected diagnostic marker in the sample taken after administration ofthe therapeutic as compared to the level of the selected diagnosticmarker in the sample taken before administration of the therapeuticagent is indicative of a positive effect of the therapeutic agent on AMDor atherosclerosis in the subject.

[0041] II. Diagnostic Kits

[0042] Diagnostic kits and reagents which may be employed in assays todetect the presence of a CEP adduct in bodily samples of test subjectsare provided. The diagnostic kit comprises an antibody, preferably amonoclonal antibody, which is used in an immunoassay to detect thepresence or quantify the amount of CEP adduct present in a sample.Preferably, the diagnostic kit further comprises a CEP adduct such as,for example, an adduct in which the CEP adduct is conjugated to aprotein or peptide. Such adduct may be employed to generate a standardcurve for quantification or as a competitor to demonstrate antibodyspecificity.

EXAMPLES

[0043] The following examples are for purposes of illustration only andare not intended to limit the scope of the claims which are appendedhereto. All references cited herein are specifically incorporated hereinby reference.

[0044] Abbreviations:

[0045] AA, arachidonic acid; AA-PC, 2-arachidonylphosphatidylcholine;Ac-Gly-Lys-OMe, methyl 6-amino-2-((acetylamino)acetyl)amino)hexanoate;BCA, bicinchoninic acid; BHT, butylated hydroxytoluene; BRB,blood-retinal barrier; BSA, bovine serum albumin; CAP,2-(carboxyalkyl)pyrrole; CEO, chicken egg ovalbumin; CEP,2-(ω-carboxyethyl)pyrrole; CHP, 2-(ω-carboxyheptyl)pyrrole; CPP,2-(ω-carboxypropyl)pyrrole; DHA, docosahexaenoic acid; DODA,9,12-dioxododecanoic acid; DOHA, 4,7-dioxoheptanoic acid; DOHA-dipep,8-(1-(5-((acetylamino)acetyl)amino)-5-(methoxycarbonyl)pentyl)pyrrol-2-yl)hexanoicacid; DOOA, 5,8-dioxooctanoic acid; EDTA, ethylenediaminetetraacetate;EI, electron ionization; ELISA, enzyme-linked immunosorbent assay; HNE,(E)-4-hydroxy-2-nonenal; HODA, 9-hydroxy-12-oxo-10-dodecenoic acid;HOHA, (E)-4-hydroxy-7-oxohept-5-enoic acid; HOOA,5-hydroxy-8-oxo-6-octenoic acid; HRMS, high resolution mass spectrum;HSA, human serum albumin; IgG, immunoglobin G; KLH, keyhole limpethemocyanin; LA, linoleic acid; LA-PC, 2-linoleylphosphatidylcholine;LDL, low density lipoprotein; LSC, liquid scintillation counting; MDA,malondialdehyde; m/z, mass to charge ratio; NMR, nuclear magneticresonance; ON, 4-oxononanal; PBS, phosphate buffered saline; PC,phosphatidylcholine; PLA₂, phospholipase A₂; PP-ACA, 2-pentylpyrrolated6-aminocaproic acid; PUFA, polyunsaturated fatty acid; R_(f), retentionfactor; ROS, rod outer segment; RPE, retinal pigment epithelium;TBDMSCl, t-butyl(dimethyl)silyl chloride; THF, tetrahydrofuran; TLC,thin layer chromatography.

[0046] Oxidation of Polyunsaturated Fatty Acids

[0047] We recently showed that oxidation of polyunsaturated fatty acids(PUFAs) in the presence of protein leads to the generation of2-(ω-carboxyalkyl)pyrrole (CAP) epitopes (FIG. 1) (Fliesler, S. J. andAnderson, R. E., (1983) Chemistry and metabolism of lipids in thevertebrate retina, Prog. Lipid Res. 22 79-131). Thus, oxidativefragmentation of arachidonic acid (AA) or linoleic acid (LA) produces5-hydroxy-8-oxooct-6-enoic acid (HOOA) or 9-hydroxy-12-oxododec-10-enoicacid (HODA), respectively, and these products are capable of reactingwith protein to generate protein-bound 2-(ω-carboxypropyl)pyrrole (CPPs)or 2-(ω-carboxyheptyl)pyrroles (CHPs). Oxidative fragmentation ofseveral other common PUFAs can also produce CHPs and CPPs. In contrast,only one common PUFA, DHA, is expected to give rise to CEPs by oxidativecleavage to HOHA (see FIG. 1). Tissues rich in DHA-containing lipidsinclude the retina and certain regions of the brain. DHA lipids arefound at significantly lower levels in blood plasma and most othertissues.

[0048] To selectively detect the occurrence of CEP adducts in vivo usingimmunological tools, it is highly desirable to raise antibodies that candistinguish between CEP adducts and CPP adducts, adducts that differ byonly one CH₂, group. As indicated below, we have determined that (i)remarkably high structural discrimination can be achieved with rabbitpolyclonal antibodies, (ii) both oxidation of a DHA phospholipid in thepresence of protein and reaction of a synthetic phospholipid ester ofHOHA with protein generates CEP esters, and (iii) CEP immuno-reactivityis abundant in DHA-rich regions of the retina.

Example 1

[0049] Peptide and Protein-Based CEPs and Rabbit Anti-CEP Antibody

[0050] A. Methods

[0051] Methyl 6-(2,5-Dioxolanyl)-4-oxohexanoate (1Me). A solution of2-(2-bromoethyl)-1,3-dioxolane (4.37 g, 24 mmol) in anhydroustetrahydrofuran (THF, 25 ml) was added dropwise over 1.5 h to Mgturnings (0.6 g, 24.7 mmol) under argon, while maintaining thetemperature below 35° C. (Boga, C., Savoia, D., Trombini, C. andUmani-Ronchi, A. (1986), A short route to2-(6-methoxycarbonylhexyl)cycoplent-2-en-1-one, Synthesis, 212-213). Thereaction mixture was then left at room temperature overnight. TheGrignard reaction mixture was cooled to −78° C., and3-carbomethoxypropionyl chloride (3.1 g, 20.5 mmol) dissolved dry THF(20 mL) was added dropwise over 1 h. The resulting mixture was thenstirred for 20 min, quenched with a saturated aqueous solution of NH₄Cl(50 mL), and extracted with EtOAc (4×50 mL). The combined organic phasewas washed with brine, dried with MgSO₄, and evaporated to obtain thecrude product. The crude compound was purified by silica gelchromatography (EtOAc/hexane, 3:7, v/v, as eluant) to yield 2.3 g (45%)of pure keto ester 1Me. ¹H NMR (200 MHz, CDCl₃). δ 4.91 (t, J=4.3 Hz,1H), 3.83-3.96 (m, 4H), 3.73 (s, 3H), 2.75 (t, J=7.4 Hz, 2H), 2.59 (m,4H); 1.98 (m, 2H); ¹³C NMR (CDCl₃) δ 208.02 (CO), 173.29 (CO), 103.22(CH), 65.00 (CH₂), 51.82 (CH₃), 37.03 (CH₂), 36.44 (CH₂), 27.74 (CH₂),27.53 (CH₂). HRMS (EI) (m/z) calcd for C₁₀H₁₅O₅ (M⁺-H) 215.0996, found215.0919.

[0052] 6-(2,5-Dioxolanyl)-4-oxohexanoic Acid (1H). Ester 1Me (396.5 mg,1.8 mmol) in 10 mL of H₂O/MeOH/THF (2:5:3, v/v/v) was stirred for 1.5 hwith NaOH (366.7 mg, 9.2 mmol) at room temperature (Kaur, K. Salomon, R.G., O'Neil, J. and Hoff, H. F. (1997) (Carboxyalkyl)pyrroles in humanplasma and oxidized low-density lipoproteins, Chem Res. Toxicol 10,1387-1396). The reaction mixture was then acidified to pH 3.0 andextracted with EtOAc (3×25 mL). The combined organic phase was washedwith H₂O, and then EtOAc was concentrated by rotary evaporation to giveacid acetal 1H (304 mg, 82%). ¹H NMR (200 MHz, CDCl₃) δ 4.91 (t, J=4.2Hz, 1H), 3.80-3.99 (m, 4H), 2.62-2.74 (m, 4H), 2.58 (t, J=7.4 Hz, 2H);1.99 (q, J=7.4 Hz, 4.2 Hz, 2H); ¹³C NMR (CDCl₃) δ 208.02 (CO), 178.47(CO), 103.18 (CH), 64.98 (CH₂), 36.78 (CH₂), 36.35 (CH₂), 27.81 (CH₂),27.48 (CH₂). HRMS (EI) (m/z) calcd for C₉H₁₄₀₅ (M⁺) 202.0814, found202.0868, calcd for C₉H₁₃O₄ (M⁺-OH) 185.0814, found 185.0818.

[0053] 4,7-Dioxoheptanoic Acid (DOHA, 2H). The acid acetal 1H (304 mg,1.5 mmol) was stirred in acetone (55 mL) and H₂O (3-5 drops) withAmberlyst catalyst (2.3 g) at room temperature for 5 h (Rees, M. S., vanKuijk, F. G. J. M., Siakotos, A. N. and Mundy, B. P., (1995) Improvedsynthesis of various isotope labeled 4-hydroxyalkenals and peroxidationintermediates, Synth. Commun. 25, 3225-3236). The reaction mixture wasfiltered through a bed of anhydrous MgSO₄. The solvent was removed byrotary evaporation to obtain the keto aldehyde 2H (220 mg) that was usedwithout further purification to prepare carboxyethyl pyrroles. ¹H NMR(CDCl₃) δ 2.43 (m, 4H), 2.77 (m, 4H), 9.70 and 9.60 (1H) ¹³C NMR (CDCl₃)208.31 (CO), 202.02 (CO), 173.82 (CO), 36.62 (CH₂), 36.54 (CH₂), 34.38(CH₂), 27.41 (CH₂). DOHA (2H) was characterized further by conversioninto a pyrrole derivative, DOHA-dipep (3H, vide infra).

[0054]8-(1-(5-((2-(Acetylamino)acetyl)amino)-5-(methoxycarbonyl)pentyl)pyrrol-2-yl)hexanoicAcid (DOHA-dipep, 3H). DOHA (2 mg, 0.01 mmol) and methyl6-amino-2-((2-acetylamino)acetyl)amino)hexanoate (Ac-Gly-Lys-OMe, 3.7mg, 0.012 mmol) dissolved in MeOH (0.3 mL) were stirred for 48 h undernitrogen at room temperature. Solvents were then removed by evaporationinto a dry ice-cooled trap using high vacuum. The crude product waspurified by chromatography on silica gel with CHCl₃/MeOH (9:1, v/v) aseluant to deliver the title CEP derivative DOHA-dipep (3H): ¹H NMR(CD₃OD) δ 1.36 (m, 2H), 1.72 (m, 2H), 1.86 (m, 2H), 2.06 (s, 3H), 2.59(t, J=7.4, 2H), 2.75 (t, J=7.8, 2H), 3.70 (s, 3H), 3.89 (m, 4H), 4.41(m, 1H), 5.80 (m, 1H), 5.93 (m, 1H), 6.58 (m, 1H); HRMS calcd forC₁₈H₂₇N₃O₆ (M⁺) 381.1900, found 381.1895.

[0055] CEP-KLH Antigen. DOHA (2H, 2.4 mg) and KLH (4.67 mg) in 0.5 M pH7.4 sodium phosphate buffer (1 mL) was incubated at room temperatureunder argon for 4 days followed by three successive 12 h dialysesagainst 10 mM pH 7.4 sodium phosphate buffer (3×1 L). The final proteinconcentration (2.72 mg/mL) was determined using the Pierce bicinchoninicacid (BCA) protein reagent (Smith, P. K., Krohn, R. I., Hermanson, G.T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K.,Goeke, N. M., Olson, B. J. and Klenk, D.C. (1985) Measurements ofprotein using bicinchoninic acid. Anal. Biochem. 150, 76-85). Thus, aseries of standard solutions of BSA (0.2, 0.4, 0.6, 0.8, 1.0, and 1.2mg/mL) were prepared. One-tenth and one-fifth dilutions (100 μL each) ofsample (CEP-KLH) were prepared. Equal volumes (2000 μL) of BCA proteinassay solution were added to the standards (100 μL) and samples (100μL). The resulting mixtures were then vortexed and incubated at 37° C.for 2 h. The pyrrole concentration (46 μM) was determined using Ehrlichreagent, 4-(dimethylamino)benzaldehyde, as described previously (15)using DOHA-dipep (3H) as a standard.

[0056] CEP-HSA. A solution of DOHA (2H, 56 mg, containing about 50% ofthe ω-ketoaldehyde form) and HSA (0.08 mM), final concentration, in 7 mLof 0.5 M pH 7.4 sodium phosphate buffer was incubated at roomtemperature under argon for 10 days, and then dialyzed twice (24 h each)against 1 L of 10 mM sodium phosphate buffer (pH 7.4). The final proteinconcentration determined using Pierce BCA protein assay, as describedabove, was 3.8 mg/mL. The pyrrole concentration (87 μM) was determinedusing Ehrlich reagent, 4-(dimethylamino)benzaldehyde, as describedpreviously (DeCaprio, A. P., Jackowshi, S. J. and Regan, K. A., (1987)Mechanism of formation and quantitation of imines, pyrroles, and stablenonpyrrole adducts in 2,5-hexanedione-treated protein, Mol. Pharmacol.32, 542-548) using DOHA-dipep (3H) as a standard. This corresponds to apyrrole:HSA ratio of 1.5:1. The presence of lysine-based CEP in CEP-HSA,the DOHA adduct of HSA, was demonstrated by nanoelectrospray massspectrometry and MS/MS analysis of tryptic peptides.

[0057] Tryptic Digestion of CEP-HSA. CEP-HSA (3.8 mg/mL) was diluted to1 mg/mL with water. Urea and ammonium bicarbonate were added to make thefinal concentrations 8 M for Urea, and 400 mM for ammonium bicarbonate.Reduction of disulfide bonds was achieved by treatment with DTT (10 mMfinal concentration). After 30 min at room temperature, the thiol groupswere alkylated by the addition of iodoacetamide (50 mM finalconcentration) and incubation for an additional 30 min at roomtemperature. After dialysis against 10 mM ammonium bicarbonate (4×500mL) for 24 hours, 20 μL of this sample (0.87 mg/mL) was then digested at37° C. with modified trypsin (Promega), added in 2 equal portions (0.1μg per addition) at 12 h intervals.

[0058] MALDI-TOF Mass Spectrometric Analysis of CEP-HSA TrypticPeptides. MALDI-TOF analysis was done using a PE Biosystems Voyager DEPro instrument equipped with a nitrogen laser (337 nm) and operated inthe delayed extraction and reflector mode with a matrix ofω-cyano-4-hydroxycinnamic acid (5 mg/mL in acetonitrile/water/3%trifluoroacetic acid, 5:4:1, v/v/v). Internal standards were used forcalibration, which included two synthetic peptides, G91 (MH+ 1015.579)and L20R (MH+ 2474.630). One μL of sample was mixed with 1 μL of matrixand 0.5 μL of internal standard mixture. Two μL of the resulting mixturewas applied to the sample plate and allowed to dry. Each spectrum wasaccumulated for ˜250 laser shots. Measured peptide masses were used tosearch the Swiss Prot, TrEMBL or NCBI sequence databases for proteinidentification. Using MS-Fit(http://prospector.ucsf.edu/htmlucsf3.0/msfit.htm), the peptide mapdataset was searched with a mass tolerance of 0.005% error (=50 ppm).

[0059] Characterization of CEP Modified Tryptic Peptides. Peptides withputative CEP modifications were analyzed by tandem nanoelectrosprayMS/MS spectrometry, using a PE Sciex API 3000 triple quadrupoleelectrospray instrument fitted with a nanospray interface (Protana).Tryptic digests were eluted from Zip Tips (Millipore) in 80%acetonitrile/20% water containing 0.02% trifluoroacetic acid, and 5 μLsamples were infused at 50 nL/min through gold coated glass capillaries(2 μm id, New Objectives, Inc). Low energy collision MS/MS was performedwith 1000-1500 V applied to the capillary, an orifice potential of 40 V,nitrogen as the collision gas, CAD gas at ˜2.1×10¹⁵ molecules/cm², and80 scans were accumulated. Spectra were acquired in positive ion modeusing a step size of 0.2 Da and 0.5 ms dwell time. The Q1 resolution waslowered to allow transmission of M+1 isotopic precursor ions into Q2; Q3was kept at unit mass resolution.

[0060] CEP-BSA. A solution of DOHA (2H, 16 mg) and BSA (0.08 mM), finalconcentration, in 0.5 M pH 7.4 sodium phosphate buffer (5 mL) wasincubated at room temperature under argon for 10 days and then dialyzedtwice (24 h each) against 10 mM pH 7.4 sodium phosphate buffer (2×1 L).The final protein concentration determined using Pierce BCA proteinassay, as described above, was 4.3 mg/mL. The pyrrole concentration (93μM) was determined using Ehrlich reagent, 4-(dimethylamino)benzaldehyde,as described previously (15) using DOHA-dipep as a standard. Thiscorresponds to a pyrrole:BSA ratio of 1.4:1.

[0061] CEP-GPDH. A solution of DOHA (2H, 30 mg) and GPDH (0.1 mM) in 5mL of 0.5 M pH 7.4 PBS was incubated at room temperature PBS (pH 7.4).The final protein concentration determined using Pierce BCA proteinassay, as described above, was 2.8 μg/ml. The pyrrole concentration (88μM) was determined using Ehrlich reagent, 4-(dimethylamino)benaldehyde,as described previously (DeCaprio, A. P., Jackowshi, S. J. and Regan, K.A., (1987) Mechanism of formation and quantitation of imines, pyrroles,and stable nonpyrrole adducts in 2,5-hexanedione-treated protein, Mol.Pharmacol. 32, 542-548) using DOHA-dipep as a standard. This correspondsto a pyrrole: GPDH ratio of 1.1:1.

[0062] Immunization. The immunogen, CEP-KLH (0.02 μmol of pyrrolegroups/mg of KLH, 2.72 mg/mL KLH in pH 7.4 PBS), was emulsified inFreund's complete adjuvant (400 μL). One Pasturella-free, New Zealandwhite rabbit was inoculated intradermally into several sites on the back(125 μL) and rear legs (125 μL). Booster injections were given every 21days. Antibody titers were monitored 10 days after each inoculation byenzyme-linked immunosorbent assay (ELISA) as described below.

[0063] Antibody Purification: Protein G Column. The crude anti-CEP-KLHantibody serum from the 92 day bleeding of the rabbit contained 18.8mg/mL protein as determined by absorbance (A₂₈₀=1.35 mg/mL) at 280 nm(Harlow, E. and Lane, D. (1988) Antibodies: a laboratory mannual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). A column was packedwith 1.0 mL of immunopure (G) immobilized protein G gel and wasequilibrated with 5 column volumes of immunopure (G) IgG binding buffer.Binding buffer (pH 5.0, 1 mL) was added to the above crude antibodyserum (1.0 mL) and the mixture was vortexed for 1 min. Then it wascentrifuged for 20 min at 3000 rpm (1900 g), and the supernatant wasapplied to the equilibrated column. The sample was allowed to flowcompletely into the gel, and 6-10 column volumes of the binding bufferwere passed through the column until the A₂₈₀ of the effluent approachedbaseline to remove the unbound serum (non-IgG proteins). The bound IgGwas then eluted with immunopure IgG elution buffer (pH 2.6) and wascollected into 1.0 mL fractions. The higher absorbance fractions werecombined and dialyzed against pH 7.4 PBS (2×2 L) at 4° C. for 24 h. Theresulting solution of anti-CEP-KLH (4 mL) contained 0.30 mg/mL purifiedIgG, as determined by absorbance at 280 nm.

[0064] Antibody Titer. To determine anti-CEP-KLH antibody level inrabbit blood serum, the BSA conjugate (CEP-BSA, containing 1.2:1pyrrole:protein molar ratio) was used as coating agent. The CEP-BSA (100μL of solution containing 8.6 μg/mL in pH 7.4 PBS) was added to eachwell of a sterilized Baxter ELISA plate. The plate was then incubated at37° C. for 1 h in a moist chamber. The coating solution was discarded.Each well was washed with PBS (3×300 μL), then filled with 1.0% chickenegg ovalbumin (CEO) in PBS (300 μL), and incubated at 37° C. for 1 h toblock remaining active sites on the plastic phase. Each well was thenwashed with 0.1% CEO in PBS (300 μL). Then rabbit serum from eachbleeding (100 μL) diluted 1:10000 with 0.2% CEO in PBS, or 0.2% CEO inPBS without serum for a black, was dispensed into the sample wells.Normal rabbit (not injected with antigen) serum diluted as above wasemployed as a negative response control. The remaining ELISA was done asdescribed previously for similar studies with anti-CPP-KLH (DOOH-KLH)and anti-CHP-KLH (DODA-KLH) antibodies (Kaur, K., Salomon, R. G.,O'Neil, J. and Hoff, H. F. (1997) (Carboxyalkyl)pyrroles in human plasmaand oxidized low-density lipoproteins. Chem Res Toxicol 10 1387-96.).The anti-CEP antibody titer rose rapidly after the second inoculationplateauing after 30 days. The rabbit was exsanguinated after 92 days anda total of 5 inoculations.

[0065] Competitive Antibody Binding Inhibition Studies. For antibodybinding inhibition studies to measure cross-reactivities, CEP-BSA,CEP-GDPH and CPP-BSA were used as coating agents and CEP-HSA and CPP-HSAwere used as standards for purified anti-CPP-KLH and anti-CPP-KLH,respectively. The ELISAs were done as described previously for studieswith anti-CPP-KLH (DOOH-KLH) antibodies (Kaur, K., Salomon, R. G.,O'Neil, J. and Hoff, H. F. (1997) (Carboxyalkyl)pyrroles in human plasmaand oxidized low-density lipoproteins. Chem Res Toxicol 10 1387-96.).Typical inhibition curves are presented in FIGS. 3 and 4.

[0066] B. Results

[0067] Synthesis of ω-Carboxyethylpyrrolated Protein and PeptideDerivatives. Paal-Knorr reactions of μ-dicarbonyl compounds with primaryamines provides an efficient route to 2-(ω-carboxyalkyl)pyrroles (Kaur,K., Salomon, R. G., O'Neil, J. and Hoff, H. F. (1997)(Carboxyalkyl)pyrroles in human plasma and oxidized low-densitylipoproteins. Chem Res Toxicol 10 1387-96.). (See FIG. 14). To generatethe requisite 2-(ω-carboxyethyl)pyrroles by this chemistry, a synthesisof the ω-ketoaldehyde DOHA (2H) was developed that exploits theselective reaction with acyl halides of the Grignard reagent producedfrom 2-(2-bromoethyl)-1,3-dioxolane. Acylation of this organomagnesiumderivative with 3-carbomethoxypropionyl chloride delivered ketoester1ME. Hydrolysis of the ethylene ketal in the derived keto acid 1Hproduced a mixture containing the requisite ω-ketoaldehyde DOHA (2H),presumably in equilibrium with the corresponding hemiacylal because the¹H NMR exhibits two aldehydic hydrogen singlets. Paal-Knorr condensationof this DOHA preparation with the dipeptide Ac-Gly-Lys-OMe provided thecarboxyethylpyrrole, DOHA-dipep (3H), that was fully characterized by ¹HNMR spectroscopy and high resolution mass spectrometry. Similar reactionof keyhole limpet hemocyanin (KLH), BSA, GPDH, or HSA deliveredcarboxyethylpyrrolated (CEP) proteins CEP-KLH, CEP-BSA, CEP-GPDH, orCEP-HSA respectively.

[0068] Mass Spectroscopic Characterization of CEP-HSA. Confirmation thatCEP-protein adducts contain carboxyethylpyrrolated lysyl residues wasaccomplished by mass spectroscopic analysis of tryptic peptides (FIG.5). Two peptides from CEP-HSA were identified by MALDI-TOF massspectroscopy, m/z 1141.6302 and m/z 2021.0678, that are apparently CEPderivatives (FIG. 5, panel A). The nanoelectrospray MS/MS spectrum ofthe m/z 1141 peptide exhibited a series of fragment ions that allowunambiguous identification of a CEP modification on the HSA lysineresidue K236 (FIG. 5, panel B). The MS/MS spectrum of the m/z 2021peptide (FIG. 5, panel C), that contains lysine residue K183, was alsoconsistent with a CEP modification.

[0069] Antibody Specificity. The structural specificities ofanti-CEP-KLH and anti-CPP-KLH antibodies were compared and contrasted(Table 2). The CEP and CPP epitopes that are, respectively, the haptensin the corresponding antigens differ by only a single CH₂ group.Nevertheless, inhibition of anti-CEP-KLH antibody binding by CPP-HSA isremarkably weak, i.e., 0.1% cross-reactivity. Even though inhibition ofanti-CPP-KLH antibody binding by CEP-HSA is somewhat greater, theanti-CPP-KLH antibodies also exhibit a high structural specificity,i.e., 1.3% cross-reactivity. Neither antibody shows significantcross-reactivity with CHP-HSA, a pyrrolated protein which contains amuch larger carboxyalkyl group than the haptens against which either ofthe antibodies were raised. The pyrrolated protein, PP-6-ACA-BSA, whichcontains an n-pentyl rather than a carboxyalkyl sidechain, showed nocross-reactivity with either of the antibodies. TABLE 2 Selectivity ofrabbit polyclonal anti-CEP-KLH and anti-CPP-KLH antibody anti-CEP-KLHantibody anti-CPP-KLH antibody CEP-BSA Coating CEP-GPDH Coating (CPP-BSACoating) IC50 %Cross- IC50 %Cross- IC50 %Cross- (pmol/well) reactivity(pmol/well) reactivity (pmol/well) reactivity

0.02 100 0.38 100 12 1.3

19 0.1 1511 0.02 0.15 100

24 0.1 802 0.05 62 0.24

N.D. 0 4097 0.01 N.D. 0

[0070] Similar structural selectivity of anti-CEP-KLH antibody wasobserved when a different coating agent, CEP-GPDH, was used (FIG. 3B).The cross-reactivity of CPP-HSA and CHP-HSA was even lower with theCEP-GDPH coating agent than with the CEP-BSA coating agent, i.e., 0.02%and 0.05% respectively. As expected, GPDH itself was not recognized byanti-CEP-KLH antibody.

Example 2

[0071] Proposed Mechanism for Production of CEP-Protein Adduct in Vitro

[0072] To test the hypothesis that protein-bound CEPs can be generatedby the reaction of HOHA-phospholipids with proteins, we prepared anester (HOHA-PC) of HOHA with 2-lyso-phosphatidylcholine (PC).

[0073] Generation of CEP Immunoreactivity by the Reaction of HOHA-PCwith HSA. Reaction of HOHA-PC with HSA results in generation ofimmunoreactivity toward anti-CEP-KLH antibodies. However, the inhibitioncurves do not parallel that for the CEP-HSA standard (FIG. 6A). This ispresumably because the CEP epitopes generated are mostly in the form ofPC esters rather than free acids against which the antibodies wereraised. If the reaction product mixture is treated with KOH, esterhydrolysis converts CEP esters into the corresponding free acids whoseinhibition curves parallel that for CEP-HSA (FIG. 6B). The yield of CEPepitope was 0.5% after 24 h based on phospholipid (Table 3). TABLE 3Generation of CEP epitope in the reaction of HOHA-PC with HSA. TimeYield (h) (pmol/ml) (%) 1 740 0.04 2 1760 0.09 4 1900 0.1 8 5200 0.3 248820 0.5

[0074] Generation of CEP Immunoreactivity by Oxidation of DHA or DHA-PCin the Presence of HSA. Oxidation of DHA for 72 h in the presence of HSAresulted in the generation of a barely detectable level (0.4 pmol/mL) ofCEP immunoreactivity that corresponds to only 7×10⁻⁵% tield based on DHA(Table 4). Similar oxidation of AA for 72 h in the presence of HSAresulted in the generation of CPP immunoreactivity that corresponds to40×10⁻⁵% yield based on AA. Generation of cross-reacting epitopes waslower or undetectable. A somewhat higher yield (0.02%) of carboxyalkylpyrrole immunoreactivity was generated upon oxidation of LA in thepresence of HSA. TABLE 4 In vitro oxidation (72 h) polyunsaturated fattyacids (PUFAs) in the presence of HSA. Immunoreactivity Antibody PUFApmol/mL (yield) Hapten Anti-CEP-KLH DHA  0.4 (7 × 10⁻⁵%) CEPAnti-CPP-KLH AA  2.7 (40 × 10⁻⁵%) CPP Anti-CHP-KLH LA  142 (0.02%) CHP

[0075] Oxidation of DHA-PC for 24 h in the presence of HSA resulted inthe generation of barely detectable CEP immunoreactivity (FIG. 7). Sincethe PC ester of CEP epitopes is not expected to be recognized byanti-CEP-KLH antibodies, the low level of immunoreactivity observedpresumably results from hydrolysis of a small portion of the ester. Asexpected, much higher levels (4×10⁻⁴% yield after 24 h oxidation) of CEPimmunoreactivity were generated if the product from oxidation of DHA-PCin the presence of HSA was treated with KOH to hydrolyze PC esters tothe corresponding free acids. Thus, DHA-containing lipids areoxidatively cleaved to HOHA-containing lipids and which react withprimary amino groups (e.g., in proteins to produce CEPs, mostly as CEPesters).

Example 3

[0076] CEP Epitopes in Retina. We used rodent retina as a test tissue toevaluate the pattern of immuno-labeling with anti-CEP-KLH antibodiesthat detect CEP epitopes. This tissue was chosen since the retina in avariety of species is known to be rich in DHA (Alvarez, R. A., et al.,Invest Ophthalmol Vis Sci 35 402-8; Wang, N. and Anderson, R. E. (1992)Curr Eye Res 11:783-91.). DHA is not uniformly distributed throughoutthe retina but, rather, is concentrated in the light-sensitivephotoreceptor rod outer segment (ROS) membranes and in the retinalpigment epithelium (RPE). DHA in these locations exists within anenvironment where photo-excitation occurs and oxygen levels and are mostabundant, i.e., at the photoreceptor-RPE interface (Koutz, C. A., et al.(1995) Effect of dietary fat on the response of the rat retina tochronic and acute light stress. Exp. Eye Res. 60 307-16.).

[0077] Immunohistochemical staining of retina from mouse by theanti-CEP-KLH antibody is intense in the photoreceptor/retinal pigmentedepithelial complex (FIG. 8). Lighter staining is also evident in theinner plexiform layer (IPL). In the photoreceptor layer, the outersegments (OS) are intensely stained while the photoreceptor innersegments (IS) are unlabeled. In contrast, when the anti-CEP-KLH antibodywas preincubated with CEP-protein antigen, all labeling was eliminated.

Example 4

[0078] CEP Immunoreactivity in Plasma of Subjects with AMD.

[0079] Human plasma obtained from patients diagnosed with AMD and normalcontrols was prepared as described previously (Salomon, R. G., Batyreva,E., Kaur, K., Sprecher, D. L., Schreiber, M. J., Crabb, J. W., Penn, M.S., DiCorletoe, A. M., Hazen, S. L. and Podrez, E. A. (2000)Isolevuglandin-protein adducts in humans: products of freeradical-induced lipid oxidation through the isoprostane pathway. BiochimBiophys Acta 1485 225-35). A metal chelator Na₂EDTA, and the antioxidantbutylated hydroxytoluenl (BHT) were used to prevent artifacts generatedby in vitro oxidation, and a cocktail of protease inhibitors was addedto prevent protein degradation. All plasma samples were quench frozenimmediately in liquid nitrogen.

[0080] The levels of CEP-protein adducts in human blood were measured byELISA using polyclonal anti-CEP antibody. We examined the plasma of (i)19 patients with diagnosed AMD of average age 82 years, designated AMD,(ii) 19 volunteers of average age 83 years who were diagnosed not tohave AMD, designated N (83), (iii) 9 young healthy volunteers of averageage 27 years, designated N (27) (FIG. 10). Comparison of levels ofCEP-protein adducts in each group is presented in FIG. 10, and the meanlevels of CEP-protein adducts (+/−S.D.) are summarized in Table 5. Theresults of statistical analyses are shown in Table 6.

[0081] The mean CEP adduct level is relatively higher in the plasma ofAMD patients (15.9 pmol/ml) than in the plasma of younger or oldervolunteers (10.5 pmol/ml) who do not have documented AMD. Independentt-test analysis of the data showed that the difference of mean CEPimmunoreactivity between AMD and both older (N83) and younger controls(N27) is significant (p=0.004 between AMD and N(83), p=0.05 between AMDand N(27)). No significant difference was found in the plasma of olderversus younger controls (p=0.97). TABLE 5 Levels (mean +/− S.D.) of CEPprotein adducts in plasma AMD N (83) N (27) Population 19 19 9 Ages 8283 27 +/− 7 CEP-protein (pmol/mL) 15.9 +/− 7.4 10.5 +/− 2.8 10.5 +/− 3.7

[0082] TABLE 6 Two tailed P-values for independent student t-testbetween all 3 groups of individuals for CEP immunoreactivity. AMD (19)Aged NL (19) Younger NL (9) AMD (19) — 0.004 0.05 Aged NL (19) 0.004 —0.97

[0083] A typical inhibition curve for binding of anti-CEP-KLH with anepitope in plasma of a patient with documented AMD, and a normal controlis shown in FIG. 9. Noteworthy is the similarity of slope compared tothe CEP-HSA standard, which supports the presumption that the epitope inhuman plasma is the same as that in the CEP-HSA. The binding inhibitionsobserved correspond to 32 pmol/mL (AMD), and 13 pmol/mL (Normal) of CEPimmunoreactivity.

Example 6

[0084] Detection of Anti-CEP Adduct Antibodies in Plasma from Patientswith AMD

[0085] A. Materials and Methods

[0086] A 96 well plate was coated with CEP-BSA (100 μL). As a blank, BSA(2%, 100 μL) was used. The plate was incubated for 1 h at 37° C., washedwith PBS (10 mM, 300 μL) 3 times and blocked with CEO (1%, 300 μL) for 1h at 37° C. Then, the plate was washed once with 0.1% chicken eggovalbum (CEO) plus 0.05% Tween 20 (300 μL). The plate was loaded withplasma, diluted 20 times with 0.2% CEO plus 0.05% Tween 20, andincubated for 1 h at room temperature. The plate was washed 3 times with0.1% CEO plus 0.05% Tween 20 (300 μL). Secondary antibodies were added(alkaline phosphatase conjugated goat anti-human IgG or alkalinephosphatase conjugated goat anti-human IgM, diluted 1:4000 with 1% CEOplus 0.05% Tween 20 (100 μL). The plate was washed 3 times with 0.1% CEOplus 0.05% Tween 20 (300 μL). A solution of disodium nitrophenylphosphate (10 mg) in glycine buffer (11 mL, 50 mM, pH 9.6) with MgCl₂ (1mM) was added. After 60 min, the absorbance was read at 405 nm withreference at 650 μm. The titre was defined as the ratio of plasmabinding to antigen (A) vs. binding to BSA (A_(o)).

[0087] B. Results

[0088] ELISA was used to study, and quantify the generation ofautoantibodies against CEP-derived protein adducts in human plasma. Inthese studies, CEP-BSA was used as antigen to coat the 96-wellmicroplate, and BSA was used as non-specific control. The titre wasdefined as the ratio of the same plasma binding to CEP-modified BSAversus native BSA. Data obtained from triplicate or quadruplicate assaysare summarized in Table 7. Plasma from 19 AMD patients showed an averagetitre of 3.4±3.1 for IgG autoantibodies binding the CEP-protein antigen,whereas, the average titre in 19 older volunteers, N(83), was 1.5±0.4which is more than 50% lower compared with the AMD cohort (Table 7).Statistical analysis (student t-test) revealed that the differencebetween these two data sets is significant (P=0.02). High titre wasassigned as any A/A_(o) value greater than 2 S.D. above the averagetitre of N(83), which means A/A_(o)>2.3. Therefore, the prevalence ofhigh anti-CEP autoantibody titres in AMD patients was 53% (10/19)compared with 21% (4/19) of the age-matched controls, N(83). TABLE 7Levels of anti-CEP autoantibodies in AMD and age-matched controls(N(82)) # of people with % of Average A/A_(o) > or = 2.3 people withPopulation A/A_(o) ± SD (Average N(83) + 2SD) A/A_(o) > 2.3 AMD 19 3.4 ±3.1 10 53 N(83) 19 1.5 ± 0.4 4 21

Example 7

[0089] Level of CEP Adducts in Subjects with Atherosclerosis.

[0090] In 7-mL vacutubes (purple top) containing EDTA (10.5 mg), bloodwas collected from ten (10) patients diagnosed with atherosclerosis andfrom healthy volunteers (N(27), and N(82)). Cells were removed bycentrifugation at 2500 rpm (1300 g) for 30 min. After transfer of thesupernatant to plastic vials, butylated hydroxytoluene (BHT; 1 mg/mL),and protease inhibitors, leupeptin (35 μM), pepstatin (5 μM), andaprotinin (0.1 TIV/mL), were added. These vials were quench-frozen byplacing in liquid nitrogen for 1 min and then stored at −80° C. untiluse.

[0091] ELISA of plasma from the 10 atherosclerosis patients and healthyvolunteers was performed as described above in Example 4. A dilutionfactor of 0.2 was employed. The results are shown in FIG. 13 and Tables8 and 9 below. TABLE 8 Mean levels (±SD) of CEP immunoreactivity inplasma Atherosclerosis Normal (83) Normal (27) Average of age (year) 5883 27 CEP (pmol/mL) 40.1 ± 23.5 10.5 ± 2.8 10.5 ± 3.7

[0092] TABLE 9 Two tail of P-values for independent student t-testbetween all 3 groups of individuals for CEP immunoreactivity.Atherosclerosis Normal (83) Normal (27) AS — 4.6e−06 0.001 N (82)4.6e−06 — 0.97

[0093] In view of the relatively low levels of DHA-containing lipids inthe blood, it is surprising that the mean CEP adduct level is muchhigher in the plasma of atherosclerosis patients (40.1 pmol/ml) than inthe plasma of aged or younger volunteers (10.5 pmol/ml) who do not havedocumented AS. Independent t-test analysis of the data showed that theaverage plasma CEP immunoreactivity is significantly different betweenpatients (atherosclerosis) and non-patient controls (N83 and N27)).(p=4.6e−06 between atherosclerosis and N(83), p=0.001 betweenatherosclerosis and N(27). This high difference in CEP epitope levelsfound in the blood of patients with atherosclerosis compared to healthyvolunteers indicates that CEP-based tests are useful for detectingatherosclerosis in patients suspected of having cardiovascular disease.

Example 8

[0094] Preparation, Characterization, and Applications of MonoclonalAnti-CEP-KLH Antibodies

[0095] Monoclonal antibodies immunospecific for CEP adducts wereproduced in collaboration with the the Hydrodoma Core Facility at theCleveland Clinic Foundation. A CEP-KLH antigen was used to obtaintwenty-eight different single cell clones producing monoclonalanti-CEP-KLH antibodies that were identified and tested further byWestern blot analysis against CEP-HSA as a standard protein containingthe CEP modification. Five clones exhibiting the strongest apparentimmunoreactivity were grown in larger amounts. Monoclonal anti-CEP-KLHantibody from these clones was purified essentially as described for thepolyclonal anti-CEP-KLH antibody, except Protein A columns were usedinstead of Protein G columns. The protein concentrations of the purifiedmonoclonal anti-bodies were determined by the Bradford method (M. M.Bradford, Anal Biochem 72, 248-54 (May 7, 1976) and Western analysisperformed with control proteins using methods and chemiluminescencedetection described elsewhere (J. W. Crabb, et al., J Biol Chem 26616674-83 (Sep. 5, 1991), B. N. Kennedy, et al., J Biol Chem 273, 5591-8(Mar. 6, 1998)). Comprehensive characterization of the specificities oftwo of these antibodies, designated anti-CEP monoclonal antibody 3 (inAb3) and anti-CEP monoclonal antibody 4 (mAb4), was pursued.

[0096] A. Preparation and Characterization of Monoclonal Anti-CEPAntibodies

[0097] Immunization. Four Balb/c mice were used for monoclonal antibodyproduction. Two mice received intraperitoneal (IP) injections of theimmunogen (CEP-KLH) and the remaining two received subcutaneous (Sub-Q)injections of the same immunogen. All mice were ear tagged andidentified by number.

[0098] Initial injection—the immunogen preparation was mixed with anequivalent volume of Complete Fruend's Adjuvant (Sigma, F-5881, CFA).For the IP injections, 0.25 ml the antigenic solution was emulsifiedwith 0.25 ml CFA. The mouse received 0.5 ml of the emulsion utilizing a25 g needle. The animals received their second injection two weeks afterthe first injection. The second injections were given utilizing the sameroute and volume as the initial injection. Incomplete Fruend's Adjuvant(Sigma, F-5506, IFA) was used for this and all subsequent injections.Three weeks latter a third injection was given.

[0099] Ten to fourteen days following the third injection a blood samplewas collected from each mouse. The mice were anesthetized with Halothanein the CCF animal facility. When the mice showed no response to the footpinch test, they were restrained by hand and the retro orbital sinus waspunctured with the tip of a 250 μl non-heparinized capillary tube(Fisher cat# 02-668-10). Approximately 200 μl of blood was collectedfrom each mouse. After the bleed the mice were returned to theirrespective cages to recover.

[0100] Serum was prepared and tested from each blood sample. The titerof the sera was determined whether the mouse received any additionalimmunizations or bleeds. If the titer was not satisfactory the mouse wasreinjected as described above and bled after 10-14 days. Once asatisfactory titer was established from any 1 of the 4 mice, a fusionwas initiated.

[0101] To prepare for the fusion, three weeks after the last IPinjection, the mouse with the highest titer was injected intravenously(IV) with 0.1 ml of the sterile antigenic solution without adjuvantutilizing a 27 g needle. The mouse was restrained with a small rodentrestraint for this injection.

[0102] Three days post IV injection the mouse was anesthetized and bledas previously described. After the bleeding, the mouse was euthanizedvia Halothane inhalation and the spleen asceptically removed.

[0103] Fusion protocol. All fusion work was done in a cell culturelaminar flow hood and under sterile conditions. A suspension of myelomascells (Sp2/0-Ag14 cells, ATCC cat# CRL-1581) to spleen cells at a ratioof 1:1 to 1:2 was prepared. The myeloma:spleen cell suspension wascentrifuged and the supernatant carefully aspirated off of the mixedpellet. 1 ml of the 50% PEG solution added to the pellet over a oneminute period. After the PEG was added, the tube was incubated in a 37°C. water bath for one minute. After the one minute incubation, 1.0 ml ofDMEM+PS was added to the tube over one minute then 15 ml of DMEM+PS wasadded over a three minute period. After the media was added, the tubewas centrifuged described above. The final pellet was resuspended inDMEM+PS+10% FBS+1×2-mercaptoethanol (Sigma, cat# M-7522, 100×=0.035%)+1×Hypoxanthine-Thymidine (Gibco, cat# 11067-030, 100×HT)+10% HybridomaCloning Factor (Origin, HCF) at a concentration of 8×10⁵ originalmyeloma cells per ml of media. As a control, 2.0 ml of a suspension ofunfused myeloma cells was prepared at 8×10⁵ cells per ml in the samemedia. The fused cell suspension (100 μl) was added to the inner 60wells in 96 well plates (Falcon 353072). The unfused myeloma cellsuspension (100 μl) was added to a minimum of 12 wells. Approximately300 ul of sterile milliQ water was added to the outer most wells. Plateswere incubated for 24 hours at 37° C. with 10% CO₂ and humidity. After24 hours, 100 ul of DMEM+PS+10% FBS+1×ME+1×HT+2× Aminopterin (Sigma,cat# A5159, 50×A)+10% HCF was added to all wells including the controlwells with unfused myeloma cells. Every three days 50 ul of this mediawas added to the fusion wells.

[0104] Clonal Selection. At approximately 5 days post fusion the initialsigns of viable hybridoma colonies were observable in the wells. Inconjunction the wells containing unfused myeloma cells were dead ordying. By 12-14 days post fusion the hybridoma colonies weremacroscopically visible and ready for assay by ELISA for antibodycontaining wells. ELISA evaluation of over 1500 wells was performedessentially as described for polyclonal antibody titer but usinganti-mouse IgG as the secondary antibody. Positive wells producinganti-CEP antibody were selected by limiting dilution cloning inDMEM+PS+10% FBS+10% HCF. Approximately 4 days after cloning, each wellwas examined to identify colonies derived from a single cell.

[0105] B. Specificity of Monoclonal Antibodies.

[0106] To assess the structural specificity and selectivity of themonoclonal anti-CEP-KLH antibodies, mAb3 and mAb4, competitiveinhibition of antibody binding to CEP-modified proteins by varioushaptens was examined. For all cross-reactivity studies, CEP-BSA was usedas coating agent and CEP-HSA was used as a standard. The concentrationat 50% inhibition (IC₅₀) for CEP-HSA was defined as 100%cross-reactivity. Duplicates of serial dilutions of all inhibitors wereused and final curves were constructed using mean absorbance values. ThemAb4 binds more strongly with the CEP-BSA. Its IC₅₀ (1.989 pmol/well) isabout 4 times less than that of mAb3 (IC₅₀=7.661 pmol/well).

[0107] For various pyrrole modifications of the HSA, both mAb3 and mAb4show remarkable selectivity (Table 10). When the carboxyalkyl side chainis extended from two to three CH₂ groups, i.e., from CEP-HSA to CPP-HSA,a cross-reactivity of only 0.3% is found with mAb4, and 0.2% with mAb3.Also, CHP-HSA which has an even longer side chain, i.e. with 7 CH₂groups and a carboxylic acid at the end, exhibited a cross-reactivity of0.3% with both antibodies. PP-HSA, that lacks the carboxyl group presentin the CEP hapten against which these antibodies were raised, displayedlittle affinity for both antibodies. HSA itself was not recognized bythese antibodies at all. TABLE 10 Specificity of Monoclonal Antibodies.CEP- CHP- CPP- PP- HSA HSA HSA HSA HSA mAb4 IC₅₀ 1.989 628 407 2621114e+8 % Crossreactivity 100 0.3 0.3 7e−4 ND mAb3 IC₅₀ 7.661 3028 2367249061 4e+8 % Crossreactivity 100 0.2 0.3 3e−3 ND

[0108] C. CEP Immunoreactivity in Mouse Retina

[0109] Mouse tissues were obtained from BALB/C mice after CO₂asphyxiation and cervical dislocation. Eyes were gently opened with acut through the eye wall with a razor blade posterior to the limbus, andwere immersion fixed in a solution of 4% formaldehyde (freshly preparedfrom paraformaldehyde), in phosphate buffer (0.1 M, pH 7.2). Afterovernight fixation, tissues were rinsed in several changes of PBS andprocessed for paraffin microscopy using conventional procedures.Sections, cut at 3 μm, were placed on Superfrost slides. Sections weredeparaffinized with xylene and hydrated through graded ethanols.

[0110] Tissue sections were incubated with 6% BSA in phosphate buffer(0.1 M, pH 7.2) for 30 min to block non-specific binding, then incubatedfor 16 h at 4° C. with anti-CEP antibody, i.e., anti-DOHA-KLH (dilutedin 6% BSA/PBS 1:100). For control sections, anti-CEP antibody waspre-incubated with CEP-HSA or CPP-HSA. After washing extensively withPBS, sections were treated with goat-anti-rabbit IgG conjugated toperoxidase ABC (Vector Labs, Burlingame, Calif., 1:200 dilution) for 1 hat room temperature. Sections were washed with PBS and incubated in0.05% DAB (3,3′-diaminobenzidine, Sigma Chemical Co., St. Louis, Mo.)and 0.03% hydrogen peroxide in the phosphate buffer at room temperature.Sections were viewed without counterstaining using DIC microscopy. Theimages presented were digitized with a Hamamatsu CCD camera, manipulatedin Adobe Photoshop and assembled in Microsoft PowerPoint.

[0111] Immunoreactivity in Mouse Retina. Immunostaining of mouse retinausing monoclonal antibody, mAb4, revealed the presence and localizationof CEP adducts. As found previously with polycolnal rabbit anti-CEP-KLHantibody, CEP immunoreactivity was prominent in the RPE andphotoreceptor outer segment (POS), where DHA is most abundant and oxygenlevels are high. However, no CEP adducts were detected in other areas ofretina, in contrast to immunostaining of rat retina by the rabbitpolyclonal anti-CEP-KLH antibody, which exhibited less selectiveimmunostaining.

What is claimed is:
 1. A method for diagnosing a disease associated withoxidation of docosahexaenoic acid (DHA) containing lipids in a testsubject, comprising: determining the level of 2-(ω-carboxyethyl) pyrrole(CEP) adducts in a bodily fluid obtained from the test subject, whereinthe presence of an elevated level of CEP adducts in the test subject'sbodily fluid as compared to the level of CEP adducts found in acorresponding bodily fluid obtained from control subjects indicates thatthe test subject has a disease associated with oxidative damage totissue comprising DHA containing lipids.
 2. The method of claim 1wherein the bodily fluid is whole blood, serum, or plasma.
 3. The methodof claim 1 wherein the disease is age-related macular degeneration(AMD).
 4. The method of claim 1 wherein the disease is AMD and the levelof CEP adducts in the bodily fluid are determined by contacting thebodily fluid with an anti-CEP antibody and assaying for the formation ofan antigen-antibody complex between the anti-CEP antibody and a moleculein the bodily fluid.
 5. The method of claim 1 wherein an antioxidant, aprotease inhibitor, or both have been added to the bodily fluidfollowing isolation of the bodily fluid from the test subject.
 6. Themethod of claim 1 wherein the disease is AMD and wherein the levels ofCEP adducts in the bodily fluid is compared to a predetermined valueobtained by determining the level of CEP adducts in a correspondingbodily fluid obtained from healthy subjects.
 7. The method of claim 1wherein the disease is AMD and wherein the levels of CEP adducts aredetermined in a plurality of bodily fluids obtained at successive timeintervals from the test subject.
 8. The method of claim 1 wherein thedisease is AMD and wherein the assay comprises determining the levels ofCEP adducts in a bodily fluid obtained from the subject at a time priorto treatment with an anti-AMD drug and determining the levels of the CEPadducts in a corresponding bodily fluid obtained from the subject at atime following treatment with the anti-AMD drug.
 9. The method of claim4 wherein the antibody is a monoclonal antibody.
 10. The method of claim4 wherein the antibody has more than 100 fold greater affinity for a CEPadduct than a 2-(ω-carboxypropyl) pyrrole (CPP) adduct.
 11. The methodof claim 1 wherein the disease is atherosclerosis.
 12. A method forcharacterizing a test subjects predisposition to developing age-relatedmacular degeneration or atherosclerosis, comprising: determining thelevel of CEP adducts in a bodily fluid obtained from the test subject,wherein the presence of an elevated level of CEP adducts in the testsubject's bodily fluid as compared to the level of CEP adducts found ina corresponding bodily fluid obtained from control subjects indicatesthat the test subject has AMD or atherosclerosis or a predisposition todeveloping AMD or atherosclerosis.
 13. The method of claim 12 whereinthe bodily fluid is whole blood, serum, or plasma.
 14. The method ofclaim 12 wherein the level of CEP adducts in the bodily fluid aredetermined by contacting the bodily fluid with an anti-CEP antibody andassaying for the formation of an antigen-antibody complex between saidantibody and a molecule in the bodily fluid.
 15. The method of claim 12wherein an antioxidant, protease inhibitor, or both have been added tothe bodily fluid following isolation after the bodily fluid from thetest subject.
 16. A method of identifying a subject who has or is atrisk of developing a disease associated with oxidation of DHA-containinglipids, comprising determining the level of anti-CEP antibodies in abodily fluid obtained from the test subject, wherein the presence of anelevated level of anti-CEP antibodies in the test subject's bodily fluidas compared to the level of anti-CEP antibodies found in a correspondingbodily fluid obtained from control subjects indicates that the testsubject has a disease associated with oxidation of DHA-containinglipids.
 17. The method of claim 16 wherein the subject is suspected ofhaving AMD or a predisposition to developing AMD.
 18. The method ofclaim 16 wherein the subject is suspected of having atherosclerosis or apredisposition to developing atherosclerosis.
 19. A diagnostic kit fordiagnosing diseases which involve oxidation of DHA, comprising: anantibody reactive with a CEP adduct,
 20. The diagnostic kit of claim 18wherein said antibody is a monoclonal antibody.
 21. The diagnostic kitof claim 19 further comprising a synthetic peptide or a syntheticprotein conjugated to CEP.
 22. The diagnostic kit of claim 21 furthercomprising a synthetic peptide or a synthetic protein conjugated to a2-(ω-carboxyalkyl) pyrrole (CAP) other than CEP.
 23. A CEP adductcomprising CEP conjugated to a carrier with a primary amino group. 24.The CEP adducts of claim 23, wherein the carrier is a peptide orprotein.